Comparison of Multiscale Imaging Methods for Brain Research

A major challenge in neuroscience is how to study structural alterations in the brain. Even small changes in synaptic composition could have severe outcomes for body functions. Many neuropathological diseases are attributable to disorganization of particular synaptic proteins. Yet, to detect and comprehensively describe and evaluate such often rather subtle deviations from the normal physiological status in a detailed and quantitative manner is very challenging. Here, we have compared side-by-side several commercially available light microscopes for their suitability in visualizing synaptic components in larger parts of the brain at low resolution, at extended resolution as well as at super-resolution. Microscopic technologies included stereo, widefield, deconvolution, confocal, and super-resolution set-ups. We also analyzed the impact of adaptive optics, a motorized objective correction collar and CUDA graphics card technology on imaging quality and acquisition speed. Our observations evaluate a basic set of techniques, which allow for multi-color brain imaging from centimeter to nanometer scales. The comparative multi-modal strategy we established can be used as a guide for researchers to select the most appropriate light microscopy method in addressing specific questions in brain research, and we also give insights into recent developments such as optical aberration corrections

Characterization of small molecule inhibitors of the AKAP-Lbc-induced RhoA activation

Das A-kinase Ankerprotein AKAP-Lbc verankert die Proteinkinase A (PKA) und fungiert als Guaninnukleotidaustauschfaktor (GEF) für die kleine GTPase RhoA. AKAP-Lbc vermittelt als Multienzymkomplex die α-Adrenozeptor-(AR-)-induzierte Hypertrophie in Kardiomyozyten. Um die Funktion des AKAP-Lbc/RhoA-Komplexes dabei näher zu charakterisieren, wurde eine Methode entwickelt, die als Hochdurchsatzverfahren zur Suche nach möglichen Inhibitoren der AKAP-Lbc- katalysierten Aktivierung von RhoA verwendet werden kann. Als Grundlage wurde die GEF-Aktivität des AKAP-Lbc, exakter der Nukleotidaustausch von GDP zu GTP von RhoA, genutzt, welcher mittels dem fluoreszenzmarkierten GTP-Analogon 2'(3')-O-(N-methylanthraniloyl)-Guanosintriphosphat (mGTP) visualisiert wurde. Die für ein High-throughput Screening benötigten Mengen an reinen, aktiven Proteinen wurden rekombinant in Bakterienzellen exprimiert, aus dem Bakterienlysat isoliert und mittels Affinitätschromatographie gereinigt. Eine Bibliothek von 18.000 niedermolekularen Substanzen wurde in einem verlässlichen Hochdurchsatzverfahren nach potenziellen Inhibitoren durchsucht. Nach dem Validierungsprozess hemmten drei Substanzen (31413, 31864 und 31892) den Austausch von GDP-RhoA zu GTP-RhoA, und damit die Aktivierung von RhoA in vitro. Die ermittelten IC50-Werte lagen bei 25 µM (31413), 40 µM (31864) und 79 µM (31892). Aufgrund der strukturellen Ähnlichkeit wurden auch bekannte Furocoumarine auf ihre Wirkung auf den AKAP-Lbc-vermittelten Nukleotidaustausches von RhoA untersucht. Keine von diesen Substanzen zeigte im verwendeten Konzentrationsbereich eine Hemmung. Untersuchungen der Verträglichkeit an Zellen wiesen darauf hin, dass die Substanzen in den verwendeten Konzentrationen nicht toxisch waren. Dafür hemmte die Substanz 31413 die Lysophosphatidsäure-induzierte Stressfaserbildung in 3T3-Zellen sowie die Gα12- aber nicht Gαq-vermittelte RhoA-Aktivierung in HEK293-Zellen. Ferner zeigte die Substanz 31413 nur eine Hemmung der AKAP-Lbc-vermittelten, SRE-induzierten Luziferase-Aktivität. Diese Ergebnisse belegten, dass 31413 eine spezifische Hemmung der AKAP-Lbc-vermittelten Rho-Aktivierung hervorruft. Mit Blick auf die Wechselwirkung zwischen AKAP-Lbc und RhoA und deren Beteiligung an der α-adrenerg-induzierten Herzmuskelhypertrophie, wurden Experimente an isolierten, neonatalen Kardiomyozyten angeschlossen. Dabei wurde für die Substanz 31413 eine Hemmung der α-AR-induzierten, nicht jedoch der α-AR-stimulierten Erhöhung der Schlagfrequenz von neonatalen Kardiomyozyten ermittelt. Die Beobachtung, dass 31413 die Adrenalin-induzierte Kontraktion nicht hemmt, ist demnach ein weiterer Beleg für die spezifische Wirkung des Moleküls auf den α-AR-induzierten RhoA-Signalweg. Dies impliziert, dass auch die α-AR-ausgelöste Hypertrophie beeinflusst werden könnte. Damit stellt die identifizierte Substanz ein wegweisendes Werkzeug für die Entwicklung neuer Therapiemöglichkeiten für Herzhypertrophie und der daraus hervorgehenden Herzinsuffizienz dar.The A kinase anchoring protein AKAP-Lbc tethers protein kinase A (PKA) and possesses a guanine nucleotide exchange factor (GEF) activity which stimulates the small GTP-binding protein RhoA. RhoA controls a plethora of cellular processes including gene expression, cellular growth and cytoskeletal dynamics. Pathological changes of its function, in particular with regard to cytoskeletal structures are often responses to chronic stress signals, a common characteristic of many diseases including chronic heart failure. In order to analyse the functional relevance of the AKAP-Lbc/RhoA signalling complex in such processes small molecule inhibitors of the AKAP-Lbc-induced RhoA activation should be identified. AKAP-Lbc catalyzes the exchange of GDP to GTP in RhoA promoting its activation. Thus a nucleotide exchange assay for screening a library of about 18,000 small molecules was established using recombinant proteins and the fluorescently-labelled GTP analogue 2'(3')-O-(N-methylanthraniloyl)-Guanosintriphosphat (mGTP). Three compounds (31413, 31864 and 31892) were found to inhibit the AKAP-Lbc-mediated nucleotide exchange from GDP-bound RhoA to GTP-bound RhoA and, consequently, RhoA activation in vitro. Measured IC50 values were 25 µM (31413), 40 µM (31864) or 79 µM (31892). Compound 31413 inhibits stress fiber formation induced by lysophosphatidic acid in 3T3 cells. For 31413 an inhibition of Gα12-mediated, but not Gαq-induced activation of RhoA was observed in HEK293 cells. Further, 31413 seemed to specifically inhibit AKAP-Lbc-mediated SRE- induced luciferase activation. These results imply that 31413 specifically blocks AKAP-Lbc-mediated RhoA activation. Bearing in mind the participation of the interaction between AKAP-Lbc and RhoA in the development of cardiac hypertrophy, experiments with isolated neonatal cardiac myocytes were conducted. Compound 31413 inhibits α1-adrenergic increase in beating frequency of isolated neonatal cardiac myocytes. This observation implies that α1-adrenergic induced cardiac hypertrophy may be modulated by the compound. This small molecule represents not only a valuable tool, but may pave the way to novel approaches for the treatment of diseases that involve AKAP-Lbc- mediated activation of RhoA such as chronic heart failure

A-kinase anchoring proteins as potential drug targets

A-kinase anchoring proteins (AKAPs) crucially contribute to the spatial and temporal control of cellular signalling. They directly interact with a variety of protein binding partners and cellular constituents, thereby directing pools of signalling components to defined locales. In particular, AKAPs mediate compartmentalization of cAMP signalling. Alterations in AKAP expression and their interactions are associated with or cause diseases including chronic heart failure, various cancers and disorders of the immune system such as HIV. A number of cellular dysfunctions result from mutations of specific AKAPs. The link between malfunctions of single AKAP complexes and a disease makes AKAPs and their interactions interesting targets for the development of novel drugs

Language Modelling for the Clinical Semantic Verbal Fluency Task

Semantic Verbal Fluency (SVF) tests are common neuropsychological tasks, in which patients are asked to name as many words belonging to a semantic category as they can in 60 seconds. These tests are sensitive to even early forms of dementia caused by e.g. Alzheimer's disease. Performance is usually measured as the total number of correct responses. Clinical research has shown that not only the raw count, but also production strategy is a relevant clinical marker. We employed language modelling (LM) as a natural technique to model production in this task. Comparing different LMs, we show that perplexity of a persons SVF production predicts dementia well (F1 = 0.83). Demented patients show significantly lower perplexity, thus are more predictable. Persons in advanced stages of de-mentia differ in predictability of word choice and production strategy-people in early stages only in predictability of production strategy

Patients with amnestic MCI Fail to Adapt Executive Control When Repeatedly Tested with Semantic Verbal Fluency Tasks

Objective: Semantic verbal fluency (SVF) tasks require individuals to name items from a specified category within a fixed time. An impaired SVF performance is well documented in patients with amnestic Mild Cognitive Impairment (aMCI). The two leading theoretical views suggest either loss of semantic knowledge or impaired executive control to be responsible. Method: We assessed SVF 3 times on 2 consecutive days in 29 healthy controls (HC) and 29 patients with aMCI with the aim to answer the question which of the two views holds true. Results: When doing the task for the first time, patients with aMCI produced fewer and more common words with a shorter mean response latency. When tested repeatedly, only healthy volunteers increased performance. Likewise, only the performance of HC indicated two distinct retrieval processes: a prompt retrieval of readily available items at the beginning of the task and an active search through semantic space towards the end. With repeated assessment, the pool of readily available items became larger in HC, but not patients with aMCI. Conclusion: The production of fewer and more common words in aMCI points to a smaller search set and supports the loss of semantic knowledge view. The failure to improve performance as well as the lack of distinct retrieval processes point to an additional impairment in executive control. Our data did not clearly favour one theoretical view over the other, but rather indicates that the impairment of patients with aMCI in SVF is due to a combination of both

Language Modelling for the Clinical Semantic Verbal Fluency Task

International audienceSemantic Verbal Fluency (SVF) tests are common neuropsychological tasks, in which patients are asked to name as many words belonging to a semantic category as they can in 60 seconds. These tests are sensitive to even early forms of dementia caused by e.g. Alzheimer's disease. Performance is usually measured as the total number of correct responses. Clinical research has shown that not only the raw count, but also production strategy is a relevant clinical marker. We employed language modelling (LM) as a natural technique to model production in this task. Comparing different LMs, we show that perplexity of a persons SVF production predicts dementia well (F1 = 0.83). Demented patients show significantly lower perplexity, thus are more predictable. Persons in advanced stages of de-mentia differ in predictability of word choice and production strategy-people in early stages only in predictability of production strategy

Exploitation vs. exploration—computational temporal and semantic analysis explains semantic verbal fluency impairment in Alzheimer's disease

Impaired Semantic Verbal Fluency (SVF) in dementia due to Alzheimer's Disease (AD) and its precursor Mild Cognitive Impairment (MCI) is well known. Yet, it remains open whether this impairment mirrors the breakdown of semantic memory retrieval processes or executive control processes. Therefore, qualitative analysis of the SVF has been proposed but is limited in terms of methodology and feasibility in clinical practice. Consequently, research draws no conclusive picture which of these afore-mentioned processes drives the SVF impairment in AD and MCI. This study uses a qualitative computational approach—combining temporal and semantic information—to investigate exploitation and exploration patterns as indicators for semantic memory retrieval and executive control processes. Audio SVF recordings of 20 controls (C, 66–81 years), 55 MCI (57–94 years) and 20 AD subjects (66–82 years) were assessed while groups were matched according to age and education. All groups produced, on average, the same amount of semantically related items in rapid succession within word clusters. Conversely, towards AD, there was a clear decline in semantic as well as temporal exploration patterns between clusters. Results strongly point towards preserved exploitation—semantic memory retrieval processes—and hampered exploration—executive control processes—in AD and potentially in MCI

Crossing borders of material science - a new approach of aerogel preparation for electron microscopy

A new method for the embedding and preparation of organic aerogels for electron mciroscopic Analysis like Crosssectioning, Lamella preparation, FIB-Tomography and Transmissio Electron Microscop

Detecting subtle signs of depression with automated speech analysis in a non-clinical sample

Abstract Background Automated speech analysis has gained increasing attention to help diagnosing depression. Most previous studies, however, focused on comparing speech in patients with major depressive disorder to that in healthy volunteers. An alternative may be to associate speech with depressive symptoms in a non-clinical sample as this may help to find early and sensitive markers in those at risk of depression. Methods We included n = 118 healthy young adults (mean age: 23.5 ± 3.7 years; 77% women) and asked them to talk about a positive and a negative event in their life. Then, we assessed the level of depressive symptoms with a self-report questionnaire, with scores ranging from 0–60. We transcribed speech data and extracted acoustic as well as linguistic features. Then, we tested whether individuals below or above the cut-off of clinically relevant depressive symptoms differed in speech features. Next, we predicted whether someone would be below or above that cut-off as well as the individual scores on the depression questionnaire. Since depression is associated with cognitive slowing or attentional deficits, we finally correlated depression scores with performance in the Trail Making Test. Results In our sample, n = 93 individuals scored below and n = 25 scored above cut-off for clinically relevant depressive symptoms. Most speech features did not differ significantly between both groups, but individuals above cut-off spoke more than those below that cut-off in the positive and the negative story. In addition, higher depression scores in that group were associated with slower completion time of the Trail Making Test. We were able to predict with 93% accuracy who would be below or above cut-off. In addition, we were able to predict the individual depression scores with low mean absolute error (3.90), with best performance achieved by a support vector machine. Conclusions Our results indicate that even in a sample without a clinical diagnosis of depression, changes in speech relate to higher depression scores. This should be investigated in more detail in the future. In a longitudinal study, it may be tested whether speech features found in our study represent early and sensitive markers for subsequent depression in individuals at risk